title: Maximizing peptide identification events in proteomic workflows using data-dependent acquisition (DDA).
abstract: Current analytical strategies for collecting proteomic data using data-dependent acquisition (DDA) are limited by the low analytical reproducibility of the method. Proteomic discovery efforts that exploit the benefits of DDA, such as providing peptide sequence information, but that enable improved analytical reproducibility, represent an ideal scenario for maximizing measureable peptide identifications in "shotgun"-type proteomic studies. Therefore, we propose an analytical workflow combining DDA with retention time aligned extracted ion chromatogram (XIC) areas obtained from high mass accuracy MS1 data acquired in parallel. We applied this workflow to the analyses of sample matrixes prepared from mouse blood plasma and brain tissues and observed increases in peptide detection of up to 30.5% due to the comparison of peptide MS1 XIC areas following retention time alignment of co-identified peptides. Furthermore, we show that the approach is quantitative using peptide standards diluted into a complex matrix. These data revealed that peptide MS1 XIC areas provide linear response of over three orders of magnitude down to low femtomole (fmol) levels. These findings argue that augmenting "shotgun" proteomic workflows with retention time alignment of peptide identifications and comparative analyses of corresponding peptide MS1 XIC areas improve the analytical performance of global proteomic discovery methods using DDA.
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261;
authors.forename: Nicholas W
authors.initials: NW
authors.lastname: Bateman
authors.forename: Scott P
authors.initials: SP
authors.lastname: Goulding
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Shulman
authors.forename: Avinash K
authors.initials: AK
authors.lastname: Gadok
authors.forename: Karen K
authors.initials: KK
authors.lastname: Szumlinski
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.forename: Christine C
authors.initials: CC
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D051379:Mice; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D012984:Software
chemical_list: D010455:Peptides